尿素吸收型工业黄酒酵母单倍体工程菌的构建

氨基甲酸乙酯是黄酒生产中的副产物,具有致癌性。尿素是氨基甲酸乙酯(EC)的主要前体物质,为降低黄酒中EC的含量,通过提高尿素转运蛋白基因DUR3的表达水平,构建了尿素吸收型工业黄酒酵母单倍体工程菌。采用融合PCR技术,将DUR3基因置于强启动子PGK1p后,构建过表达组件"URA3-PGK1p-DUR3-PGK1tURA3",转化工业黄酒酵母单倍体Na(MATa),获得单倍体工程菌Na-uD。通过实验室黄酒发酵,工程菌所酿黄酒发酵液中尿素含量降低了55.0%,EC含量降低了10.9%,其他理化指标无明显差异。因此过表达DUR3基因的工程菌Na-uD具有"吸收尿素"的能力,能够降低发酵液中尿素的含量,且无外源抗性基因的引入。     

低产尿素黄酒酵母工程菌的酿造特性

为考察酵母工程菌在黄酒酿造过程中的发酵性能及其降低发酵液中尿素和氨基甲酸乙酯(ethyl carba-mate,EC)的能力,以前期构建的降低黄酒中尿素和EC效果最好的酵母工程菌N85^DUR1,2-c为研究对象,利用单因素试验考察黄酒发酵工艺对其降低发酵液中尿素和EC能力的影响,并对其在生产试验过程中的发酵性能进行研究。结果表明,酵母接种量、发酵温度以及麦曲添加量等工艺参数对工程菌N85^DUR1,2-c低产尿素和EC的性能没有明显的影响,且含量低于亲本菌株。50 kL生产试验表明,工程菌N85^DUR1,2-c所酿黄酒中理化指标含量正常,符合黄酒国标的要求。而N85^DUR1,2-c发酵液中尿素和EC的含量分别为(2.4±0.2)mg/L和(14.9±0.6)μg/L,较亲本菌株分别降低了90.7%和54.6%,且贮存过程中EC含量增加缓慢。说明酵母工程菌N85^DUR1,2-c在不改变黄酒优良品质的前提下,能够显著地降低发酵液中尿素的含量,可以从根源上减少黄酒中EC的积累,提高饮用...     

黄酒酵母的改良及对产物中尿素和氨基甲酸乙酯含量的影响

氨基甲酸乙酯（EC)是一种潜在致癌物,在黄酒发酵过程中尿素是它的前体物质。该研究通过紫外诱变和基因过表达筛选获得改良黄酒酵母菌种,并对黄酒产品的理化指标进行检测可知,与出发菌株相比,改造菌株的发酵性能和黄酒的出酒率、酒精度、总糖、总酸、氨基酸态氮和β-苯乙醇没有明显的差异,而诱变菌株JF501-A62发酵产物尿素含量降低了67%,EC含量降低了59%;基因过表达菌株JF501-B5发酵产物尿素含量降低了88%,EC含量降低了63%。两者均有很好的发酵性能,并取得了较好地降低产品中尿素含量、进而降低氨基甲酸乙酯含量的效果。与紫外诱变相比,基因过表达的改良方法获得了尿素含量更低的菌株,并贮存6个月之后产品中的EC含量更低。     

基于PCR方法敲除黄酒酵母精氨酸酶基因的工程菌构建

用一种高效快速的免克隆方法-长侧翼同源PCR（LFH-PCR）,构建基因敲除组件,然后通过化学转化方法把敲除组件转入黄酒酵母细胞中,利用同源重组机制精确敲除精氨酸酶基因（CAR1）,构建了黄酒酵母工程菌株。结果表明,敲除CAR1基因的工程菌与出发菌株相比,酒液中尿素的含量降低了72%,氨基甲酸乙酯含量降低了38%,并且该菌株的遗传稳定性好,发酵性能与出发菌株基本一致。      

黄酒酵母的性能改良及降低其产物中氨基甲酸乙酯含量的研究

氨基甲酸乙酯(EC)是一种潜在致癌物,在黄酒发酵过程中尿素是它的前体物质。该研究通过紫外诱变和基因过表达筛选获得改良黄酒酵母菌种,并对黄酒产品的理化指标进行检测可知,与原始菌株相比,改造菌株酿造的发酵性能和黄酒的出酒率、酒精度、总糖、总酸、氨基酸态氮没有明显的差异,而诱变菌株JF501-A62发酵产物尿素含量降低了67%,EC含量降低了59%;基因过表达菌株JF501-B5发酵产物尿素含量降低了88%,EC含量降低了63%。两者均有很好的发酵性能,并取得了较好的降低产品中尿素含量、进而降低氨基甲酸乙酯含量的效果。与紫外诱变相比,基因过表达的改良方法获得了尿素含量更低的菌株,且贮存6个月之后产品中的EC含量更低。     

CRISPR/Cas9介导的低产尿素黄酒酵母工程菌的构建

通过代谢工程改造构建低产尿素的酿酒酵母工程菌,从根源上减少黄酒发酵液中尿素的含量及氨基甲酸乙酯(ethyl carbamate,EC)的形成。该研究利用融合PCR构建DUR3过表达组件"HOL-PGK1p-DUR3-PGK1tHOR",通过CRISPR/Cas9介导的基因组编辑技术转化酿酒酵母S. cerevisiae NaDUR1,2-Δcar1,在敲除CAR1和过表达DUR1,2基因的基础上过表达DUR3基因,获得工程菌S. cerevisiae NaDUR1,2/DUR3-Δcar1。实验室黄酒发酵实验结果表明,与亲本菌株S. cerevisiae Na相比,工程菌S. cerevisiae NaDUR1,2/DUR3-Δcar1所酿黄酒发酵液中尿素含量降低了92. 1%,EC含量降低了58. 6%;与出发菌株S. cerevisiae NaDUR1,2-Δcar1相比,工程菌S. cerevisiae NaDUR1,2/DUR3-Δcar1所酿黄酒发酵液中尿素含量降低了43. 4%,EC含量降低了16. 2%。过表达DUR3的工程菌S. cerevisiae NaDUR1,2/DUR3-Δcar1具有"尿素吸收"的能力,减少EC的形成。借助CRISPR/Cas9系统,构建的酵母工程菌无外源抗性基因的引入,具有工业化应用的潜在可能性。     

黄酒酵母ADH2、ALD2和ALD6基因敲除对黄酒中乙醛代谢的影响

以工业黄酒酵母N85尿嘧啶缺陷型单倍体2a-u3(MATa ura3)为出发菌株,研究分别敲除乙醛代谢相关酶的编码基因ADH2、ALD2和ALD6对乙醛代谢的影响。采用融合PCR技术分别构建乙醇脱氢酶编码基因ADH2敲除组件"ADH2S-URA3-ADH2X"和乙醛脱氢酶编码基因ALD2和ALD6敲除组件"ALD2S-URA3-ALD2X"和"ALD6S-URA3-ALD6X",转化N85尿嘧啶缺陷型单倍体菌株后,以尿嘧啶缺陷型的恢复为筛选标记,分别获得ADH2、ALD2和ALD6基因缺失菌株2a-A2(Δadh2)、2a-D2(Δald2)和2a-D6(Δald6)。分别将获得的工程菌与亲本菌株以及前期构建的丙酮酸脱羧酶基因PDC5缺失菌株2a-P5(Δpdc5)进行实验室规模的黄酒发酵实验。结果表明,与亲本菌株2a相比,工程菌2a-A2发酵液中乙醛含量降低了7.14%,而2a-P5发酵液中乙醛含量降低了32.68%,说明工业黄酒酵母乙醛的生成存在糖代谢途径,并且乙醛主要由丙酮酸脱羧而来;工程菌2a-D2和2a-D6发酵液中乙醛含量与亲本相比分别增加了144.91%和88.87%,说明乙醛脱氢酶是黄酒中控制乙醛降解的关键酶,而且ALD2基因在乙醛转化为乙酸过程中发挥更关键的作用。     

黑曲霉葡萄糖氧化酶基因在毕赤酵母SMD1168中的表达

在毕赤酵母SMD1168中利用乙醇氧化酶AOX1强启动子表达黑曲霉葡萄糖氧化酶(Glucose oxidase,GOD)。提取黑曲霉Aspergillus niger PCTC的基因组DNA,以此为模板进行PCR扩增获得葡萄糖氧化酶基因,将目的基因插入到具有AOX1强启动子的表达载体p PICZαA上,经电转化导入毕赤酵母SMD1168中。经zeocin抗性平板初筛、摇床复筛以及SDS-PAGE蛋白质电泳的检测,获得了一株产葡萄糖氧化酶活力的菌株,该株菌在30℃、200 r/min的培养条件下,经体积分数1.0%的甲醇诱导发酵1 d可获得1.12 U/mL的酶活。对该菌株进行了摇瓶产酶条件优化,其最佳发酵条件为:在pH 5、30℃下经体积分数1.5%甲醇诱导7 d,酶活为32 U/mL。     

短乳杆菌鸟氨酸氨甲酰基转移酶对黄酒发酵中氨基甲酸乙酯的调控作用

氨基甲酸乙酯是存在于黄酒发酵过程中的一种潜在致癌物质。本文研究添加鸟氨酸氨甲酰基转移酶对氨基甲酸乙酯及黄酒基础品质的影响。根据鸟氨酸氨甲酰基转移酶可以催化、降解氨基甲酸乙酯的前体物瓜氨酸的原理,采用基因工程手段从短乳杆菌中克隆并大量表达鸟氨酸氨甲酰基转移酶,经Ni-NTA琼脂糖纯化树脂纯化后添加到黄酒发酵的不同阶段。结果表明:在发酵中期添加的鸟氨酸氨甲酰基转移酶能显著降低氨基甲酸乙酯的含量,最高可降低至原含量的15%,且对其主要前体物——尿素和瓜氨酸均有影响,同时检测黄酒的理化品质,结果表明游离氨基酸、挥发性风味物质及味觉特性差异不显著,说明添加鸟氨酸氨甲酰基转移酶未对产品性质产生影响,为工业上降低氨基甲酸乙酯的形成提供新方向。     

赖氨酸芽孢杆菌产氨基甲酸乙酯水解酶的发酵条件优化

氨基甲酸乙酯是发酵酒精饮料及发酵食品中一种广泛存在的致癌物,酶法降解是解决氨基甲酸乙酯污染的重要途径之一。作者以一株来源于小鼠胃部具有水解氨基甲酸乙酯活性的菌株Lysinibacillus fusiformis SC02为出发菌株,通过摇瓶水平单因素实验对其产酶条件进行了优化。优化后的培养基组成为:半乳糖25 g/L,大豆蛋白胨20 g/L,尿素4 g/L,硫酸铜0.02 g/L,pH6.0。最佳产酶的发酵条件为:发酵温度37℃,接种体积分数3%,装液量20 m L/250 m L。在上述优化的培养基和培养条件下,产酶水平由900 U/L提高到4 500 U/L,提高了350%。在3 L发酵罐水平上初步探究了不同搅拌转速对菌株产酶的影响。当搅拌转速达到800 r/min,菌株最高酶活水平由4 500 U/L提高到7 066 U/L,提高了57%。     

Enhancement of urea uptake in Chinese rice wine yeast strain N85 by the constitutive expression of DUR3 for ethyl carbamate elimination

Most of the fermented alcoholic beverages, particularly Chinese rice wine, contain the potentially human carcinogenic compound ethyl carbamate (EC). As a major EC precursor in Chinese rice wine, urea in fermentations can be transported into the yeast cell by urea permease and finally metabolized by urea carboxylase and allophanate hydrolase in vivo. To eliminate EC in Chinese rice wines, the present study constructed high urea uptake yeast strains N1‐D, N2‐D and N‐D, by introducing a strong promoter (PGK1p) into the urea permease gene (DUR3) of the industrial Chinese rice wine yeast N85, and by the restoration of the URA3 gene at the same time. With these self‐cloned, high urea uptake strains, the urea and EC in the terminal Chinese rice wine samples were reduced to different extents. With two copies of overexpressed DUR3, the N‐D strain could reduce the urea and the EC by 53.4 and 26.1%, respectively. No difference in fermentation characteristics was found between the engineered strains and the parental industrial yeast strain N85. These results could help to optimize the genetic manipulation strategy for EC elimination in Chinese rice wine production. Copyright © 2015 The Institute of Brewing & Distilling     

The overexpression of DUR1,2 and deletion of CAR1 in an industrial Saccharomyces cerevisiae strain and effects on nitrogen catabolite repression in Chinese rice wine production

Urea is acknowledged as the predominant precursor of ethyl carbamate (EC) in Chinese rice wine. During Chinese rice wine fermentation, urea accumulates owing to the nitrogen catabolite repression effect when preferred nitrogen sources are available. In previous research, two metabolically engineered strains were constructed with overexpression of DUR1,2 and deletion of CAR1 from an industrial Chinese rice wine yeast N85. The decreasing effect of urea and EC was demonstrated during small‐scale Chinese rice wine fermentations. The present study compared the urea utilization rate of the parental and metabolically engineered yeast strains, using a preferred and non‐preferred nitrogen source culture media, leading to alleviated urea accumulation and thus EC formation. The qRT‐PCR results showed that, in all of the culture media, DUR1,2 was overexpressed with the inserted strong promoter PGK1p. During pilot scale fermentations, the urea and EC content decreased with the engineered strains. These results confirmed that the engineered strains could resist the nitrogen catabolite repression effect. Copyright © 2016 The Institute of Brewing & Distilling     

Effect of citrulline metabolism in Saccharomyces cerevisiae on the formation of ethyl carbamate during Chinese rice wine fermentation

Urea, as the main precursor of ethyl carbamate (EC), has received extensive attention. Here, we have metabolically engineered an industrial yeast strain – Saccharomyces cerevisiae N85 – to investigate the contribution of the EC precursor citrulline to the concentration of EC in Chinese rice wine. The results showed that the citrulline biosynthetic pathway of the modified strain N85‐arg3 was completely suppressed by deletion of ARG3, encoding ornithine carbamoyltransferase. However, there were no significant differences in the levels of citrulline or EC between N85‐arg3 and the parental strain N85 during fermentation. In addition, we over‐expressed ARG1 (encoding argininosuccinate synthase) and ARG4 (encoding argininosuccinate lyase) to construct the engineered strains N85^ARG1,4 and N85^ARG1,4‐arg3. The citrulline contents in Chinese rice wine fermented with N85^ARG1,4 and N85^ARG1,4‐arg3 were respectively 24.1 and 20.4% less than that of N85. However, the contents of EC were 23.8 and 28.5% more than that of N85. These results suggested that reducing the formation of EC during Chinese rice wine fermentation by genetically engineering citrulline metabolism in S. cerevisiae was not a viable proposition. Copyright © 2018 The Institute of Brewing & Distilling     

YDL080C和LEU2基因敲除对工业黄酒酵母异戊醇生成量的影响

通过敲除生成途径中关键酶的编码基因,对异戊醇在工业黄酒酵母N85中的生成进行了研究。采用融合PCR技术,分别构建类丙酮酸脱羧酶基因（YDL080C）缺失组件\     

Citrulline production by lactic acid bacteria in Chinese rice wine

Ethyl carbamate (EC) is a carcinogenic compound derived from the spontaneous reaction of ethanol with urea or citrulline in Chinese rice wine. Polymerase chain reaction–denaturing gradient gel electrophoresis showed that five species, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis and Lactobacillus coryniformis were the most abundant bacteria in the Chinese rice wine production process. Five strains belonging to these species can degrade arginine primarily in the exponential growth phase and accumulate citrulline in MRS‐Arg medium. In addition, an L. brevis strain was shown to be capable of assimilating citrulline, indicating the potential of this strain suggesting a potential route to reduce citrulline content and ethyl carbamate formation in Chinese rice wine fermentation. Copyright © 2018 The Institute of Brewing & Distilling     

Determination of ethyl carbamate in Chinese yellow rice wine using high‐performance liquid chromatography with fluorescence detection

Ethyl carbamate (EC) is a naturally occurring component in the fermented products, especially the Chinese yellow rice wine. EC has been found showing a potential for carcinogenity and causing food safety problems. This study is to investigate the application of the existing HPLC‐FLD method to the analysis of EC in Chinese yellow rice wine, and to validate it for EC analysis with high through‐out and applicability in Chinese yellow rice wine sample. The parameters examined in this study were fully evaluated. Results indicated that good linearity was obtained with a correlation coefficient exceeding 0.9990, the limit of detection and quantification was 73.2 and 243.9 μg L^?1, respectively. Recoveries ranged between 98.30% and 101.30%, and the precision of this method was lower than 5% (RSD). The method conducted in this work was successfully applied to determine EC concentration in Chinese yellow rice wine samples from different origins. The negative correlation between EC formation and urea level in yellow rice wine samples is evaluated with the developed HPLC‐FLD method.     

Effects of sterilization temperature on the concentration of ethyl carbamate and other quality traits in Chinese rice wine

Ethyl carbamate (EC), which is present in Chinese rice wine, has a large potential for carcinogenicity and genotoxicity. EC is produced during the process of rice wine fermentation and storage. High concentrations of precursors, as well as high temperatures, will significantly accelerate the formation of EC. The present work aims to reduce EC formation by optimizing the production process, especially the boiling procedure. With various boiling sterilization temperatures, EC accumulated to different concentrations but the lower the temperature, the less EC was formed. To preserve the quality traits of Chinese rice wine, including biological and non‐biological stability, as well as the sugar component, an 80°C boiling temperature is suggested. The present study provides direction for process optimization, which combined with improved production technology and metabolic engineered yeast strains, can reduce the content of EC in Chinese rice wine. Copyright © 2014 The Institute of Brewing & Distilling